Methods and systems for sequencing long nucleic acids

Abstract

The disclosure provides methods and systems for sequencing long nucleic acid fragments. In one aspect, methods, systems and reagent kits are provided for sequencing nucleic acid target sequences. Some embodiments of the methods, systems and reagent kits are particularly suitable for sequencing a large number of fragments, particularly long fragments.

1 - 123 . (canceled) 
     
     
         124 . A method for sequencing a target nucleic acid, comprising the steps of:
 (a) hybridizing a first extension primer with the target nucleic acid;   (b) extending the first extension primer, the extending in step (b) comprising one or more first controlled extensions to a first defined length, wherein each of the one or more first controlled extensions comprises contacting the target nucleic acid with a set of nucleotides comprising three different unmodified nucleotides, wherein the extending in step (b) produces a first extended product comprising the first extension primer and a first extended portion, wherein sequence of the first extended portion is unknown; and   (c) using the first extended product obtained in step (b) as a first sequencing primer from which to generate a first sequence read, and sequencing a first region of said target nucleic acid,   with the proviso that the sequencing in (c) is not pyrophosphate detection based sequencing.   
     
     
         125 . The method of  claim 124 , further comprising:
 (d) removing the first sequence read and the first sequencing primer;   (e) hybridizing a second extension primer with the target nucleic acid;   (f) extending the second extension primer, the extending in step (f) comprising one or more second controlled extensions to a second defined length, wherein each of the one or more second controlled extensions comprising contacting the second extension primer with a second set of nucleotides comprising another three different unmodified nucleotides, wherein the extending in step (f) produces a second extended product comprising the second extension primer and a second extended portion; and   (g) using the second extended product obtained in step (f) as a second sequencing primer from which to generate a second sequence read, and sequencing a second region of said target nucleic acid.   
     
     
         126 . The method of  claim 125 , wherein the removing in (d) comprises enzymatic digestion of the first sequence read. 
     
     
         127 . The method of  claim 125 , wherein the removing in (d) comprises exonuclease digestion. 
     
     
         128 . The method of  claim 125 , wherein the first and second extended products are the same. 
     
     
         129 . The method of  claim 125 , wherein the first and second extended products are different. 
     
     
         130 . The method of  claim 124 , wherein the one or more first controlled extensions comprises (i) contacting the target nucleic acid with a first set of nucleotides comprising three different unmodified nucleotides; and (ii) contacting the target nucleic acid with a second set of nucleotides comprising three different unmodified nucleotides. 
     
     
         131 . The method of  claim 130 , further comprising: between (i) and (ii), removing the first set of nucleotides by washing, or by a nucleotide degrading enzyme. 
     
     
         132 . The method of  claim 130 , wherein the first set of nucleotides is different from the second set of nucleotides. 
     
     
         133 . The method of  claim 130 , wherein the second set of nucleotides further comprises one reversible terminator nucleotide, thereby the first extended product comprising an incorporated reversible terminator nucleotide. 
     
     
         134 . The method of  claim 133 , further comprising, in (c), deblocking the incorporated reversible terminator nucleotide before generating the first sequence read. 
     
     
         135 . The method of  claim 125 , wherein the sequence of the target nucleic acid is determined by assembling the first, second, and optionally additional sequence reads. 
     
     
         136 . The method of  claim 124 , wherein the target nucleic acid is attached to a substrate. 
     
     
         137 . The method of  claim 125 , wherein the first and second sequence reads start at positions that are at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 175, or 200 bases apart on the target nucleic acid. 
     
     
         138 . A method for sequencing a target nucleic acid, comprising the steps of:
 (a) hybridizing a first extension primer with the target nucleic acid;   (b) extending the first extension primer, the extending in step (b) comprising one or more first controlled extensions to a first defined length, wherein each of the one or more first controlled extensions comprises contacting the target nucleic acid with a set of nucleotides comprising three different unmodified nucleotides, wherein the extending in step (b) produces a first extended product comprising the first extension primer and a first extended portion, wherein sequence of the first extended portion is unknown; and   (c) using the first extended product obtained in step (b) as a first sequencing primer from which to generate a first sequence read, and sequencing a first region of said target nucleic acid,   wherein a base that is resistant to exonuclease digestion is incorporated to a position in the first sequence read, with the proviso that the sequencing in (c) is not pyrophosphate detection based sequencing.   
     
     
         139 . The method of  claim 138 , further comprising:
 (d) removing at least a part of said sequencing product.   
     
     
         140 . The method of  claim 139 , wherein the removing in (d) comprises the exonuclease digestion. 
     
     
         141 . The method of  claim 140 , further comprising:
 (e) hybridizing a second extension primer with the target nucleic acid;   (f) extending the second extension primer, the extending in step (f) comprising one or more second controlled extensions to a second defined length, wherein each of the one or more second controlled extension comprises contacting the second extension primer with a second set of nucleotides comprising another three different unmodified nucleotides, wherein the extending in step (f) produces a second extended product comprising the second extension primer and a second extended portion; and   (g) using the second extended product obtained in step (f) as a second sequencing primer from which to generate a second sequence read, and sequencing a second region of said target nucleic acid.   
     
     
         142 . The method of  claim 141 , wherein the first and second sequence reads start at positions that are at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 175, or 200 bases apart on the target nucleic acid. 
     
     
         143 . The method of  claim 141 , wherein the sequence of the target nucleic acid is determined by assembling the first, second, and optionally additional sequence reads.